Crystalline 2,5-dione-3-(1-methyl-1h-indol-3-yl)-4-[1-(pyridin-2ylmethyl)piperidin-4-yl]-1h- pyrrole mono-hydrochloride

ABSTRACT

The present invention relates to crystalline 2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-yl-methyl)piperidin-4-yl]-1H-indol-3-yl]-1H-pyrrole mono-hydrochloride salt, a pharmaceutical formulation containing said salt and to methods for treating cancer and for inhibiting tumor growth using said salt.

BACKGROUND OF THE INVENTION

Compounds of formula I:

and pharmaceutically acceptable salts thereof, useful as protein kinaseC inhibitors, were disclosed by Heath, et al., in European PatentPublication No. 817,627 (Heath).

Example #49 of Heath disclosed a free base compound of formula FB:

While FB is undoubtedly a very effective pharmaceutical agent,unexpected difficulties were encountered in its large-scale production.Thus, unpredictable formation of solvates complicated the commercialsynthesis to such an extent that it became necessary to develop analternative form for large-scale commercialization.

In this context, WO 02/02094 and WO 02/02116 specifically describe theuse of the dihydrochloride salt of FB (FB-2HCl) to treat cancer and toinhibit tumor growth as a mono-therapy or in conjunction with ananti-neoplastic agent or radiation therapy. Unfortunately, it has nowbeen determined that FB-2HCl is hygroscopic. In addition, althoughFB-2HCl appears to be crystalline by optical light microscopy, moredetailed study by X-ray powder diffraction (XRD) has revealed that thismaterial is in fact only poorly crystalline.

Surprisingly, in accordance with the invention, it has now beendiscovered that the monohydrochloride salt of FB is capable of beingreproducibly produced on a commercial scale, is not significantlyhygroscopic, is sufficiently stable for use in oral formulations, andcan be produced in a highly crystalline state.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to crystalline2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-yl]-1H-indol-3-yl]-1H-pyrrolemono-hydrochloride, a hydrate thereof, or mixtures thereof.

The present invention further relates to crystalline2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-yl]-1H-indol-3-yl]-1H-pyrrolemono-hydrochloride, a hydrate thereof, or mixtures thereof, having anX-ray diffraction pattern which comprises the following peaks: 6.8±0.1,10.9±0.1, 14.2±0.1 and 16.6±0.1° in 2θ; when the pattern is obtainedfrom a copper radiation source (CuKα; λ=1.54056 Å). This crystallinematerial is hereafter referred to as “F-I”.

The present invention also relates to a pharmaceutical compositioncontaining F-I and a pharmaceutical carrier. In another embodiment, thepharmaceutical formulation of the present invention may be adapted foruse in treating cancer and for use in inhibiting tumor growth.

Moreover, the present invention relates to methods for treating cancerand to methods for inhibiting tumor growth which comprise administeringto a mammal in need thereof an effective amount of F-I.

In addition, the present invention is related to F-I for treating cancerand for inhibiting tumor growth.

Another embodiment of the invention provides for the use of F-I for themanufacture of a medicament for the treatment of cancer and for themanufacture of a medicament for inhibiting tumor growth.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representative XRD pattern for F-I.

DETAILED DESCRIPTION OF THE INVENTION

Prior to discovering the problems associated with the large-scalemanufacturability of FB, due to a concern that FB may not possessoptimal bioavailability properties, an in situ salt screen was performedto identify salts for FB possessing improved properties. This screenevaluates the solubility of salts formed in situ in aqueous media. Thesolubility obtained in situ for a given salt is not directly predictiveof the equilibrium solubility of the crystalline form(s) of the samesalt. However, the in situ screen can be used to prioritize the saltsfor synthesis and characterization during salt selection. From thesedata, five out of seventeen mono-acid salts were chosen for synthesisand characterization. These salts were the citrate, methanesulfonate(mesylate), phosphate, tartrate and mono-hydrochloride (FB-HCl). Inaddition, FB-2HCl was also synthesized, characterized and analyzed. Someof these salts' properties as well as those of FB are discussed below.

Citrate, Mesylate, Phosphate and Tartrate

The citrate salt generated from methanol is insoluble in water. Themesylate salt is hygroscopic, exhibiting up to 2% weight gain at 70% RHand over 15% weight gain at 95% RH. Although the phosphate salt exhibitsrapid dissolution and high solubility at early time points, thesolubility of the phosphate drops to 71 μg/mL upon prolonged incubation.The phosphate salt is also somewhat hygroscopic and exhibited hysteresisin water desorption, indicating possible hydrate formation.

The tartrate is only slightly hygroscopic, exhibiting ˜1% weight gain atRH's up to 70%. Based on this and other promising initial results, thetartrate was subjected to a brief polymorph/solvate screen to determineits suitability for bulk manufacturing and use as a pharmaceutical.

The tartrate salt was initially isolated (by titration of the free basewith tartaric acid) as a crystalline hydrate. The hydrated material wasthen recrystallized to determine if other pharmaceutically relevantcrystal forms of the tartrate salt could be prepared. The number ofsolvents suitable for recrystallization was limited by the relativelypoor solubility of this salt in many solvents, including polar, proticsolvents (H₂O, methanol, ethanol and isopropyl alcohol) and manynon-protic solvents (acetone, ethyl acetate, methyl ethyl ketone andtetrahydrofuran). Sufficient solubility was observed only indimethylformamide, dimethylsulfoxide and organic (and organic/aqueous)mixtures.

Elevated temperatures were often required to achieve dissolution. Thetartrate salt was typically not generated from the recrystallizationexperiments that were carried out. Instead, a crystal form of FB wasobtained most often. A non-solvated form of the tartrate was not found.These results suggest that isolation of a tartrate salt of FB could bedifficult, presumably due to the low solubility of different crystalforms of FB relative to the tartrate salt, and the relatively smalldifference in pKa between FB and tartaric acid.

FB-2HCl

The aqueous solubility of FB-2HCl under various conditions was analyzedand at concentrations up to 10 mg/mL, solutions of FB-2HCl are stable atambient temperature for up to 10 days. However, solutions held at 50° C.exhibited profound precipitation prior to the first time point (6 days).At concentrations ≧40 mg/mL, rapid precipitation within minutes wasnoted at ambient room temperature. XRD analysis and ion chromatography(to determine chloride content) of the precipitated crystals confirmedthat this precipitate was FB-HCl.

FB

The product of the synthesis described below in Preparation 1, istypically a non-solvated crystalline form of FB. This non-solvated form(hereafter referred to as FB Form I) is preferred as it crystallizeswell in the reaction, filters rapidly and affords a high purity ofproduct (total related substances (TRS) ˜0.77%). However, under thesevery same reaction conditions, a solvate containing tetrahydrofuran(THF) is also sometimes isolated (frequency of occurrence ˜10-20%). Thiscrystalline solvate filters very slowly and traps certain impuritiesresulting in a higher TRS for product (2.42-4.78%). The high TRSassociated with this solvate has required that, when present, theisolated solvate be reworked. Despite significant research, the reasonfor the occasional formation of the solvate containing THF is unknown.The lack of control in preparation of FB Form I has limited itspotential for development as the final active pharmaceutical ingredient(API).

FB-HCl

FB-HCl, prepared via addition of 1 equivalent of concentrated or 1Nhydrochloric acid to a mixture of FB in a lower alcohol, e.g., methanol,isopropanol or 2-butanol, or in mixture of a lower alcohol and water, iscrystalline and has a melting onset temperature of about 256° C. asmeasured by differential scanning calorimetry (DSC). FB-HCl, produced asdescribed in Example 1, is relatively non-hygroscopic between 0-70% RH(<2% wt gain @ 95% RH).

Characterization of FB-HCl

Various methods, including thermogravimetric analysis (TGA), DSC and XRDwere used to characterize FB-HCl. TGA allows for measurement of theamount and rate of weight change as a function of temperature. TGA ismost commonly used to study desolvation processes and to quantitativelydetermine the total volatile content of a solid. DSC is a technique thatis often used to screen compounds for polymorphism because thetemperatures(s) at which a physical change in a material occurs isusually characteristic of that material. DSC is often used to complementTGA analysis in screening compounds for physical changes upon controlledheating. XRD is a technique that detects long-range order in acrystalline material and can be performed at different RH's to detectsubtle phase changes induced by moisture sorption.

Different lots of FB-HCl, prepared via addition of 1 equivalent ofconcentrated or 1 N hydrochloric acid to a mixture of FB in methanol,were analyzed by TGA and were found to retain different levels of water:from <0.01% (anhydrous material) all the way to 1.6% (hemi-hydrate). TheTGA results showed not only the different amounts of water present inthe crystalline FB-HCl materials, but also that the water, when present,is readily expelled from the material upon heating above ambienttemperature.

The different water contents prompted an investigation into the moisturesorption characteristics of those lots of crystalline FB-HCl that werenot anhydrous. Indeed, the various partially hydrated lots showeddistinctly different water uptake profiles. Regardless of the amount ofwater sorbed in the crystalline FB-HCl lattice, the sorption isothermsconsistently showed gradual weight gains up to ˜40% RH, above which, thewater uptake plateaued. The maximum moisture sorption (1.6% at 40% RH)observed for those partially hydrated lots of crystalline FB-HClsuggests that at full water occupancy, a hemihydrate (0.5 mole)composition is present. Crystalline FB-HCl material capable of watersorption is hereafter referred to as “hygroscopic F-I”.

The XRD peaks of hygroscopic F-I did not shift at any RH. The XRDpatterns generated for hygroscopic F-I were identical to XRD patternsgenerated for the non-hygroscopic F-I material (hereafter referred to as“anhydrous F-I”). The absence of changes to the XRD pattern when movingfrom anhydrous F-I to hygroscopic F-I, as well as the absence of changesto the XRD pattern for hygroscopic F-I as a function of humidity, showsnot only that the crystal lattice of F-I is unperturbed by the moisturesorption process, but also that moisture sorption into the particlescannot be site-specific.

F-I (both hygroscopic and anhydrous) exhibits a strong, unique XRDpattern with sharp peaks and a flat baseline, indicative of a highlycrystalline material (see FIG. 1). The angular peak positions in 2θ andcorresponding I/I_(o) data for all F-I peaks with intensities equal toor greater than 5% of the largest peak are tabulated in Table 1. Alldata in Table 1 is expressed with an accuracy of ±0.10 in 2θ. TABLE 1Angle 2θ I/I_(o) (%) 6.3 19.1 6.8 27.8 7.2 5.0 10.9 100 12.5 11.2 12.738.0 13.2 21.0 14.2 62.6 14.4 19.1 15.4 17.0 16.6 56.3 16.8 21.8 17.027.2 17.3 5.9 17.7 9.6 17.9 10.4 18.4 25.8 18.8 24.5 19.1 69.1 21.7 7.322.1 24.8 22.8 7.9 23.7 19.7 24.4 19.1 24.7 14.4 25.4 15.5 25.8 11.326.4 44.1 26.8 11.6 27.7 10.7 27.9 17.1 28.1 10.8 28.6 5.3 29.1 9.7

It is well known in the crystallography art that, for any given crystalform, the relative intensities of the diffraction peaks may vary due topreferred orientation resulting from factors such as crystal morphology.Where the effects of preferred orientation are present, peak intensitiesare altered, but the characteristic peak positions of the polymorph areunchanged. See, e.g., The United States Pharmacopeia #23, NationalFormulary #18, pages 1843-1844, 1995. Furthermore, it is also well knownin the crystallography art that, for any given crystal form, the angularpeak positions may vary slightly. For example, peak positions can shiftdue to a variation in the temperature at which a sample is analyzed,sample displacement, or the presence or absence of an internal standard.In the present case, a peak position variability of ±0.10 in 2θ willtake into account these potential variations without hindering theunequivocal identification of a crystalline salt of the presentinvention.

A well-known and accepted method for searching crystal forms in theliterature is the “Fink” method. The Fink method uses the four mostintense lines for the initial search followed by the next four mostintense lines. In general accord with the Fink method, based on peakintensities as well as peak position, F-I may be identified by thepresence of peaks at 6.8±0.1, 10.9±0.1, 14.2±0.1 and 16.6±0.1° in 2θ;when the pattern is obtained from a copper radiation source (λ=1.54056).The presence of F-I may be further verified by peaks at 6.3±0.1,7.2±0.1, 12.5±0.1, and 17.0±0.1° in 2θ; when the pattern is obtainedfrom a copper radiation source (λ=1.54056).

FB Form I vs. hygroscopic F-J vs. anhydrous F-I

Extensive equilibrium solubility determinations were undertaken for bothhygroscopic and anhydrous F-I in a variety of aqueous media at ambienttemperature. Additionally, the equilibrium solubility of FB Form I wasmeasured at ambient temperature. Samples were assayed by highperformance liquid chromatography (HPLC) after 24 hours of equilibrationin the respective solvents. The results are summarized in Table 2. TABLE2 Amt Dissolved Filtrate Sample Solvent (mg/mL) pH FB Form I 0.01 N HCl0.279, 0.355 2.20 Anhydrous F-I 0.054, 0.056 2.19 Hygroscopic F-I 0.046,0.053 2.25 FB Form I pH 2.2 buffer 0.346, 0.336 2.21 Anhydrous F-I0.360, 0.363 2.27 Hygroscopic F-I 0.324, 0.352 2.26 FB Form I SIF, fedpH 5.0 0.073, 0.074 4.94 Anhydrous F-I 0.016, 0.015 4.94 Hygroscopic F-I0.014, 0.015 4.93

The equilibrium solubility data reveal that while F-I (hygroscopic andanhydrous) and FB Form I have similar solubilities in pH 2.2 buffer, F-Iis significantly less soluble than FB Form I in 0.01N HCl and simulatedintestinal fluid (SIF) (fed). No significant differences betweenhygroscopic and anhydrous F-I in any media tested were observed.

The solubility results suggest that controlling the bulk composition(hygroscopic vs. anhydrous particles) of F-I as an API is not criticalfrom a bioavailability standpoint. To confirm that variability in thehygroscopicity of F-I lots should not adversely impact bioavailability,intrinsic dissolution rates were also measured for the hygroscopic andanhydrous F-I. For comparison purposes, the intrinsic dissolution rateof FB Form I was also measured. Because FB Form I dissolved too rapidly(>10% of a 100 mg compact dissolved within 10 minutes) and thehygroscopic and anhydrous F-I dissolved too slowly (no appreciabledissolution in 10 minutes), precise intrinsic dissolution rates couldnot be determined. The intrinsic dissolution results are summarized inTable 3. TABLE 3 % of 100 mg Compact Dissolved in 10 Minutes DissolutionMedium Hygroscopic F-I Anhydrous F-1 FB Form I 0.1 N HCl <<0.5 <<0.5 >30

The in vitro dissolution and solubility data discussed above suggestthat FB Form I should offer bioavailability advantages in vivo relativeto F-I. In order to confirm this prediction, the plasma pharmacokineticparameters of FB Form I in fed female beagle dogs were evaluatedfollowing single oral administration by gavage of 5 mg/kg of FB Form Ior F-I in a cross-over design. The 6 dogs were randomized into twotreatment groups to receive single doses of FB Form I followed by asingle dose of F-I two weeks later, or vice versa. On Days 1 and 14, 3dogs received FB Form 1 and 3 dogs received F-I, and blood samples werecollected at 0.5, 1, 2, 3, 4, 8, 12 and 24 hours post dosing.Concentrations of FB Form I were determined by liquid chromatographytandem mass spectrometry. These concentrations were subsequently used todetermine the pharmacokinetic parameters reported in Table 4. TABLE 4Cmax AUC0-24 AUC0-24 AUC0-inf AUC0-inf Dosing Cmax (nM) (nM) (nM × hr)(nM × hr) (nM × hr) (nM × hr) Animal Regimen FB Form I F-I FB Form I F-IFB Form I F-I 1 1 531.6 679.0 2092.4 2970.0 2113.9 3016.1 2 1 600.2501.4 3477.5 4064.4 3603.1 4200.8 3 1 552.4 774.1 5119.5 6150.1 5333.16630.8 4 2 717.7 450.2 2270.2 2944.5 2306.3 2998.3 5 2 336.5 481.42443.1 3592.3 2578.6 3828.6 6 2 99.8 327.4 389.3 1735.0 400.6 1798.4Dosing regimen 1 = Day 1 F-I, Day 14 FBDosing regimen 2 = Day 1 FB, Day 14 F-I

Surprisingly, based on in vitro solubility and dissolution data, plasmaexposure for F-I in terms of area under the concentration versus timecurves (AUC) for both 0 to 24 hr and 0 to infinity was significantlyhigher than that obtained from FB Form 1. Absorption rate did not appearto change as the time to reach Cmax (tmax) ranged from 1 to 2 hours forboth FB Form I and F-I. The increased exposure for F-I was most likelydue to increased bioavailability, since clearance did not appear tochange given the similarities in the apparent half-life of eliminationvalues.

Synthesis

Preparation of FB

Step 1—Stir a mixture of 2-picolyl chloride hydrochloride (7.0 g, 42.7mmol), 4-piperidone mono-hydrate hydrochloride (6.88 g, 44.8 mmol),powdered sodium carbonate (18.3 g, 173 mmol) and acetonitrile (70 mL)for 45 minutes at ambient temperature, 45 minutes at 40° C., 45 minutesat 50° C., 45 minutes at 60° C., and then heat to 70° C. with vigorousstirring. Monitor the reaction by HPLC (Zorbax RX-C8 25 cm column,acetonitrile/H₃PO₄ buffer at pH 3.0, λ=250 nm) for disappearance ofpicolyl chloride. At completion of the reaction, allow the mixture tocool to room temperature, filter to remove the insoluble solids, thenwash the filter cake with acetonitrile (2×25 ml). Concentrate thefiltrate to a small volume (˜30 ml) and solvent exchange into 41 ml ofethyl acetate. Rapidly stir and heat the solution to 55° C. then treat,over 30 minutes, with a solution of camphorsulfonic acid (9.91 g, 42.67mmol) in ethyl acetate (77 mL). Allow the resulting suspension to coolto room temperature then stir for 3 hours. Filter the precipitate, washwith ethyl acetate (2×30 ml), and dry in vacuo at 45° C. to give 15.6 g(87%) of the camphorsulfonic acid salt.

Step 2—To a 1 L 3-neck jacketed vessel under N₂, add the product of Step1 (1.0 equivalent, 33.3.g), 2-(2,2-dimethoxyethyl)aniline (Fukuyama etal, Tet. Lett., 39 (1-2):71-74, 1998; 1.0 equivalent, 14.3 g) andpropionic acid (115 mL). Stir the reaction at 20-24° C. until thecontents dissolve (15-30 minutes). Cool the mixture to −10 to −15° C.,then add 1.0 M NaBH(OPr)₃ in tetrahydrofuran (115 mL) over at least 2hours under N₂ while maintaining an internal vessel temperature <−10° C.Confirm completion of the reductive amination by HPLC (Zorbax C-8column, pH 3.0 (1.5 ml triethylamine/1.5 ml H₃PO₄/1 L H₂O. Initialgradient: 80% aqueous/20% acetonitrile. Final (45 mins): 20% aqueous/80%acetonitrile). After reaction completion is verified, add ethyl acetate(200 mL) and adjust the reaction temperature to 0° C. Adjust the pH to10.0 by careful addition of 25% NaOH (315 g) and allow the reaction towarm to 47-52° C. Stir the reaction for 30 minutes to 60 minutes at47-52° C. Stop stirring the reaction and allow the layers to settle forat least 15 minutes at 47-52° C. Remove the lower aqueous layer and washthe organic layer with aqueous 20% NaCl (150 mL). After stirring for 30minutes at 47-52° C., stop the agitation and allow the layers toseparate over 15 minutes. Remove the lower aqueous layer and reduce thereaction volume to ˜65-85 mL by vacuum distillation. Add ethyl acetate(100 mL) back to the reaction and cool the mixture to 23-25° C. Addtrifluoroacetic acid (30 ml) over at least 30 minutes. Warm the reactionto 29-31° C. and allow the reaction to proceed until by HPLC analysisthe initial amination adduct is present at less than <1.0%. Afterreaction completion is verified, add ethyl acetate (175 mL) and water(30 mL) and carefully adjust the pH to 9.0 with 25% NaOH (74 g), whilewarming to 40-45° C. Stir the resultant bi-phasic mixture for at least 1hour at 45-50° C. and allow the pH to drop to 8.60. Stop the mixing andallow the layers to settle for at least 15 minutes at 45-50° C. Removethe lower aqueous layer and wash the organic layer with aqueous 20% NaCl(125 mL) while stirring at 45-50° C. After 30 minutes stirring, and a 15minute settle time at 45-50° C., remove the aqueous layer andconcentrate the reaction mixture to 100 to 150 mL volume via vacuumdistillation. Add isopropanol (400 mL) and concentrate the reactionagain to ˜200 mL, then add additional isopropanol (200 mL). Concentratethe mixture to ˜200 mL final volume via vacuum distillation and age thesuspension for 3 hours at 43-45° C., then cool over 3-4 hours to −5° C.Filter the product at −5° C. and wash with pre-cooled (<0° C.)isopropanol (2×40 mL). Dry the reductive amination product at 50-60° C.under reduced pressure.

Step 3—Slurry the product of Step 2 (5.00 g, 17.2 mmol) with drytert-butyl methyl ether (70 mL, 14 vol.) under N₂ at 23° C. Add dryacetonitrile (20 mL, 4 volumes) at ambient temperature in one portionand heat the resulting hazy solution to 40° C. Add a solution of 2.0 MHCl in acetonitrile (8.5 mL, 17.0 mmol, 0.99 equivalent) dropwise over30 minutes while maintaining a preset jacket temperature of 40° C. Warmthe resulting slurry to 50° C. then stir for 1 hour. Cool the mixture to−10° C. over 2-3 hours. Add oxalyl chloride (2.30 mL, 26.4 mmol, 1.50equivalent) dropwise over 3-5 minutes, keeping the pot temperature <−5°C. Warm the resulting slurry to 0° C. and stir for 1-2 hours untilcomplete reaction by HPLC. Add methanol (10 mL, 2 volumes) dropwise over3-5 minutes, keeping the pot temperature <10° C. Allow the resultingslurry to gradually warm to 23° C. over 15-30 minutes then stir for 1-2hours until complete. Cool the slurry to 0-5° C., then add 2N KOH (38mL, 76 mmol, 4.4 equivalents) dropwise to adjust the pH of the mixtureto 7.8 while maintaining the vessel temperature <10° C. Stir thequenched reaction mixture at 10° C. for 15-20 minutes post pHadjustment, then remove the lower aqueous layer. Back-extract the loweraqueous layer with tert-butyl methyl ether (20 mL). Wash the combinedorganic layers (100 mL) with aqueous 20% NaCl (50 mL) for 20-30 minutesat 10° C. Allow the layers to settle for 15 minutes then remove thebrine layer. Subject the organic layer to a body feed of Na₂SO₄ (15 ganhydrous), warm to 23° C. then stir for 1-12 hours. Filter the reactionmixture then concentrate the filtrate in vacuo. Re-dissolve the residuein ethyl acetate (100 mL) then re-concentrate. Add ethyl acetate (35 mL)and CH₃CN (1 mL), heat the mixture to 45-50° C. to dissolve, then coolthe mixture to 40° C. over an hour. Optionally seed the crude mixture(30 mg) then cool to 23° C. over 2 hours after a suspension forms. Addheptane (80 mL) dropwise over 20-30 minutes to the slurry and then coolthe mixture to 0° C. over 1-2 hours. Stir the suspension for anadditional 1-2 hours at 0° C. then filter. Rinse the filter cake withcold 2:1/heptane:ethyl acetate (15 mL) then with room temperatureheptane (15 mL). Dry the filter cake in a vacuum oven at 50° C. to aconstant weight to provide 5.60 g of1-(1-[(pyridin-2-yl)methyl]piperidin-4-yl)-3-(methoxycarbonylcarbonyl)indole(87%).

Step 4—Charge a 3 neck flask equipped with an addition funnel andnitrogen purge with the product of Step 3 (10.0 grams (1.0 equivalent,26.5 mmol) and 1-methyl-3-(aminocarbonylmethyl)indole (Faul et al, J.Org. Chem., 63 (17):6053, 1998; 4.86 g, 0.975 equivalents, 25.8 mmol) intetrahydrofuran (Karl Ficher <0.03%, 72 ml, 7.2 volumes). Cool theslurry to −5 to −10° C. with an ice/acetone bath. Add potassiumt-butoxide (20% in tetrahydrofuran, 1.6 M, 36.4 ml, 2.2 equivalents,58.3 mmoles) over 10-30 minutes maintaining the reaction temperature at−10 to 5° C. Heat the reaction to 40-45° C. and stir for 1 hour togenerate a slurry. Cool the reaction to 0-10° C. with an ice/water bathand then add water (74 mL, pre-chilled to 0-10° C.) rapidly. Thereaction generally exotherms to ˜15° C. so re-cool the reaction 0-10° C.and adjust the pH to 12.7-12.9 with a mixture of concentrated HCl (5.2ml) and water (15 ml) (approximately ⅔ of this mixture is required).Adjust the pH with the remainder of the HCl/water mixture over ˜20minutes to a pH of 7.3-7.8 then stir for 30 minutes at 0-10° C. Slowlyadd water (60 mL) over 20-30 minutes at 0-10° C. and stir the reactionfor 1-2 hrs. Filter on a pressure filter and wash with a pre-chilledmixture of tetrahydrofuran (20 ml) and water (60 ml) and dry overnightat 50° C. under vacuum to give FB.

Example 1

To a 3 necked flask equipped with heating mantle, condenser anddistillate take off add FB (59.0 g, 114.4 mols), 2-butanol (949 ml, 16.1vols), deionized water (621.4 mL, 10.5 vols) and HCl (food grade: 12.24mL, 14.13 g, 0.21 volumes, 1.05 equivalents). Heat the reaction toreflux and remove half of the solvent by distillation. Slowly add2-butanol (27 volumes) over 2 hours, while maintaining a constantsolvent level in the reaction flask. Cool the reaction to roomtemperature over 60 minutes, then cool to 0-5° C. and stir for 1-2hours. Filter the product and wash the filter cake with 2 volumes of2-butanol and dry the filter cake overnight at 50° C. under vacuum togive F-I. Elemental Analysis: Theory for C₃₂H₃₀N₅O₂Cl: C, 69.62, H,5.48, N, 12.69, Cl, 6.42; Found: C, 69.29, H, 5.49, N, 12.52, Cl, 6.54.

XRD patterns were obtained on a Siemens D5000 X-ray powderdiffractometer, equipped with a CuKα source (λ=1.54056 Å) and a Kevexsolid-state detector, operating at 50 kV and 40 mA with a 1 mmdivergence and receiving slit and 0.1 mm detector slit. Each sample wasscanned between 4° and 35° in 2θ with a step size of 0.020 and a maximumscan rate of 3 sec/step. The XRD pattern for the material produced inExample 1 is as described in Table 1 and FIG. 1.

Formulation

A salt of the present invention is preferably formulated in a unitdosage form prior to administration. Therefore, yet another embodimentof the present invention is a pharmaceutical composition comprising asalt of the present invention and a pharmaceutical carrier. The term“pharmaceutical” when used herein as an adjective means substantiallynon-deleterious to the recipient patient.

The present pharmaceutical compositions are prepared by known proceduresusing well-known and readily available ingredients. In making theformulations of the present invention, the active ingredient (e.g., F-I)will usually be mixed with a carrier, or diluted by a carrier, orenclosed within a carrier that may be in the form of a capsule, sachet,paper or other container. When the carrier serves as a diluent, it maybe a solid, semisolid or liquid material that acts as a vehicle,excipient or medium for the active ingredient. Thus, the compositionscan be in the form of tablets, pills, powders, lozenges, sachets,cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol (asa solid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions and sterile packagedpowders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propylhydroxybenzoates, talc,magnesium stearate and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Thecompositions of the invention may be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient.

Formulation Example 1 25 mg Capsule

Quantity Ingredient (mg/capsule) F-I 27.1 Crospovidone XL 16.9-24.4Lactose Anhydrous 142.2-164.4 Lactose Monohydrate 142.2-164.4 MagnesiumStearate Vegetable 1.1-2.8 Povidone 13.1-16.9 Polysorbate 80 1.9-5.6

Formulation Example 2 100 ml Capsule

Quantity Ingredient (mg/capsule) F-I 108.5 Crospovidone XL 16.9-24.4Lactose Anhydrous 101.5-123.8 Lactose Monohydrate 101.5-123.8 MagnesiumStearate Vegetable 1.1-2.8 Povidone 13.1-16.9 Polysorbate 80 1.9-5.6

The capsules above are manufactured by an aqueous granulation process,as described below. The lactose, a portion of the crospovidone, and theactive ingredient (F-I) are added to the granulator and dry blended fora suitable period of time to uniformly distribute the powders. Agranulation solution consisting of povidone and polysorbate 80 inpurified water is sprayed at a uniform rate onto the powders whilemixing under specified conditions. When a suitable granulation endpointis reached, the granulator is stopped and the granulation is unloaded.

The granulation is wet sieved through a suitable screen to disrupt largeagglomerates, spread on paper lined trays, and dried in a convectionoven until the moisture is reduced to a suitable level. The size of thegranulation is reduced to a desirable range by passing through a co-millor other suitable apparatus. These sized powders are collected,transferred to a mixing apparatus, and blended with a specified quantityof magnesium stearate and additional crospovidone until uniformlydistributed. The finished powders are then filled into hard gelatincapsules either manually or on a suitable piece of automated capsulefilling equipment.

Following the filling operation, the finished capsules are visuallyinspected for external defects. To improve the pharmaceutical eleganceof the finished product, the capsules may be physically de-dusted andpolished by either manual or automated processes.

Demonstration of Function

The salt of the present invention is an inhibitor of vascularendothelial growth factor (VEGF)-induced angiogenesis. At least twoassay systems demonstrate these pharmacologic activities: 1) F-I is apotent inhibitor of VEGF-stimulated proliferation of HUVEC cells inculture upon 72 hours of exposure to the compound; 2) F-I is a highlyeffective inhibitor of VEGF-induced neo-angiogenesis in the rat cornealmicropocket when administered orally to the animals for 10 days. Theseassay systems are more fully described in WO 02/02116. The salt of thepresent invention is, thus, effective in treating cancer and inhibitingtumor growth.

Utilities

As tumor growth inhibitors, the salt of the present invention is usefulto treat cancers of the bladder, brain, breast, cervix, colorectum,esophagus, kidney, head and neck, liver, lung, ovaries, pancreas,prostate and stomach. The salt of the present invention is also usefulto treat soft tissue sarcomas and osteosarcomas and to treat Hodgkinsand non-Hodgkins lymphoma or hematological malignancies (leukemias).

Preferred methods of using a salt of the present invention relate to itsuse to treat cancers of the bladder, kidney, brain, breast, colorectum,liver, lung (non-small cell), ovaries and stomach and to its use totreat non-Hodgkins lymphoma (e.g., diffuse large B cell and mantle celllymphoma) or hematological malignancies (leukemias).

Even more preferred methods of using a salt of the present inventionrelate to its use to treat cancers of the brain, colorectum, lung(non-small cell), and to its use to treat non-Hodgkins lymphoma, β celllymphomas and β cell related leukemias.

Dose

One skilled in the art will recognize that the amount of a salt of thepresent invention to be administered in accordance with the presentinvention, that is, a therapeutically effective amount, is that amountsufficient to produce an anti-neoplastic effect, to induce apoptosis orcell death, and/or to maintain an antiangiogenic effect.

Generally, an amount of a salt of the present invention to beadministered is decided on a case-by-case basis by the attendingphysician. As a guideline, the extent and type of the neoplasia, thetiming of administration relative to other therapies (if any), and thebody weight, and age of the patient will be considered, among otherfactors, when setting an appropriate dose. Typically, an effectiveminimum daily dose of a salt of the present invention, e.g., F-I, willexceed about 200 mg (usually >400 mg, e.g., 500 mg). Usually, aneffective maximum daily dose of F-I will not exceed about 700 mg.However, in the case of glioblastomas (brain tumors) the maximum dailydose of F-I could be as high as 1400 mg. The exact glioblastoma dose maybe determined, in accordance with the standard practice in the medicalarts of “dose titrating” the recipient; that is, initially administeringa low dose of the compound, e.g., 200 or 400 mg and gradually increasingthe dose until the desired therapeutic effect is observed.

Route of Administration

The salt of the present invention can be administered by a variety ofroutes including the oral, rectal, transdermal, subcutaneous, topical,intravenous, intramuscular or intranasal routes. The oral route ispreferred.

Combination Therapy

The salt of the present invention may be used in combination withconventional anti-neoplasm therapies to treat mammals, especiallyhumans, with neoplasia. The procedures for conventional anti-neoplasmtherapies, including chemotherapies using anti-neoplastic agents andtherapeutic radiation, are readily available, and routinely practiced inthe art, e.g., see Harrison's PRINCIPLES OF INTERNAL MEDICINE 11thedition, McGraw-Hill Book Company.

Specifically, a crystalline salt of the present invention may be used toenhance the anti-neoplasm effects of an anti-neoplastic agent. A widevariety of available anti-neoplastic agents are contemplated forcombination therapy in accordance with present invention.

Anti-neoplastic agents contemplated for combination therapy inaccordance with the present invention include, but are not limited to:alkylating agents, including busulfan, chlorambucil, cyclophosphamide,ifosfamide, melphalan, nitrogen mustard, streptozocin, thiotepa, uracilnitrogen mustard, and triethylenemelamine, temozolomnide; antibioticsand plant alkaloids including actinomycin-D, bleomycin, cryptophycins,daunorubicin, doxorubicin, idarubicin, irinotecan, L-asparaginase,mitomycin-C, mithramycin, navelbine, paclitaxel, docetaxel, topotecan,vinblastine, vincristine, and VP-16; hormones and steroids includingaminoglutethimide, anastrozole, bicalutamide, DES, estramustine, ethinylestradiol, flutamide, fluoxymesterone, goserelin, hydroxyprogesterone,letrozole, leuprolide, medroxyprogesterone acetate, megestrol acetate,methyl prednisolone, methyltestosterone, mitotane, nilutamide,prednisolone, tamoxifen, testosterone and triamicnolone; syntheticsincluding all-trans retinoic acid, BCNU (carmustine), carboplatin(paraplatin), CCNU (lomustine), cis-diaminedichloroplatinum (cisplatin),dacarbazine, hexamethylmelamine, hydroxyurea, levamisole, mitoxantrone,oxaliplatin, procarbazine; antimetabolites includingchlorodeoxyadenosine, cytosine arabinoside, 2′-deoxycoformycin,fludarabine phosphate, 5-fluorouracil, 5-FUDR, gemcitabine,6-mercaptopurine, methotrexate, pemetrexed, and thioguanine; monoclonalantibodies including rituximab and trastuzumab; antisense compoundsincluding ISIS 3521; and biologics including alpha interferon, BCG,G-CSF, GM-CSF, and interleukin-2; and the like. These anti-neoplasticagents assert their cytotoxicity or anti-neoplasm effects in a varietyof specific neoplastic conditions (see WO 02/02094).

In a preferred embodiment of the invention one or more anti-neoplasticagents are selected from the group consisting of BCNU, cyclophosphamide,doxorubicin, prednisone or dexamethasone, vincristine, gemcitabine,cisplatin, 5 fluoruracil, capecitibine, CPT-11, carboplatin, paclitaxel,docetaxel, rituximab and trastuzumab.

A crystalline salt of the present invention may also be used incombination with radiation therapy. Usually, radiation is used to treatthe site of a solid tumor directly or administered by brachytherapyimplants.

Therapeutic radiation contemplated for combination therapy in accordancewith the present invention are those used in the treatment of cancerwhich include, but are not limited to X-rays, gamma radiation, highenergy electrons and High LET (Linear Energy Transfer) radiation such asprotons, neutrons, and alpha particles. The ionizing radiation isemployed by techniques well known to those skilled in the art. Forexample, X-rays and gamma rays are applied by external and/orinterstitial means from linear accelerators or radioactive sources.High-energy electrons can be produced by linear accelerators. High LETradiation is also applied from radioactive sources implantedinterstitially.

The phrase “in combination with” means that the crystalline salt of thepresent invention is administered shortly before, shortly after,concurrently, or any combination of before, after, or concurrently, withsuch other anti-neoplasm therapies. A salt of the present invention maybe administered in combination with more than one anti-neoplasm therapy.In a preferred embodiment, the a salt of the present invention isadministered from 2 weeks to 1 day before any chemotherapy, or 2 weeksto 1 day before any radiation therapy. In another preferred embodiment,a salt of the present invention may be administered duringanti-neoplastic chemotherapies and radiation therapies. If administeredfollowing such chemotherapy or radiation therapy, a salt of the presentinvention is preferably given within 1 to 14 days following the primarytreatments. A salt of the present invention may also be administeredchronically or semi-chronically, over a period of from about 2 weeks toabout 5 years.

1. Crystalline2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-yl]-1H-indol-3-yl]-1H-pyrrolemono-hydrochloride, a hydrate thereof, or mixtures thereof. 2.Crystalline2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-yl]-1H-indol-3-yl]-1H-pyrrolemono-hydrochloride, a hydrate thereof, or mixtures thereof, having anX-ray diffraction pattern which comprises the following peaks: 6.8±0.1,10.9±0.1, 14.2±0.1 and 16.6±0.1° in 2θ; when the pattern is obtainedfrom a copper radiation source (CuKα; λ=1.54056 Å).
 3. The crystallinemono-hydrochloride of claim 2 having an X-ray diffraction pattern whichfurther comprises the following peaks: 6.3±0.1, 7.2±0.1, 12.5±0.1 and17.0±0.1° in 2θ.
 4. A pharmaceutical composition comprising a salt ofclaim 1 and a pharmaceutical carrier.
 5. A method of treatingnon-Hodgkins lymphoma which comprises administering to a mammal in needthereof an effective amount of a compound of claim
 1. 6. A method oftreating glioblastoma which comprises administering to a mammal in needthereof an effective amount of a compound of claim
 1. 7. A method oftreating non-small cell lung cancer which comprises administering to amammal in need thereof an effective amount of a compound of claim 1.8-14. (canceled)
 15. A pharmaceutical composition comprising a salt ofclaim 2 and a pharmaceutical carrier.
 16. A pharmaceutical compositioncomprising a salt of claim 3 and a pharmaceutical carrier.
 17. A methodof treating non-Hodgkins lymphoma which comprises administering to amammal in need thereof an effective amount of a compound of claim
 2. 18.A method of treating non-Hodgkins lymphoma which comprises administeringto a mammal in need thereof an effective amount of a compound of claim3.
 19. A method of treating glioblastoma which comprises administeringto a mammal in need thereof an effective amount of a compound of claim2.
 20. A method of treating glioblastoma which comprises administeringto a mammal in need thereof an effective amount of a compound of claim3.
 21. A method of treating non-small cell lung cancer which comprisesadministering to a mammal in need thereof an effective amount of acompound of claim
 2. 22. A method of treating non-small cell lung cancerwhich comprises administering to a mammal in need thereof an effectiveamount of a compound of claim 3.